A compound ABAGG and uses thereof

Abstract

This invention discloses a compound ABAGG and its uses. It is produced by a Micromonospora strain that converts glycerol and glycine. The Micromonospora strain is *Micromonospora deliciosa*. Micromonospora profundus TRM 95458, deposited on August 2, 2022 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), accession number GDMCC NO:62600. This invention utilizes the described strain to produce compounds containing glycerol groups such as ABAGG, turning waste into treasure, and has good prospects for industrial application.

Description

[0001] This invention is a divisional application of Chinese Patent Application No. 202211744304.X, entitled "An Actinomycete for Converting Glycerol and Glycine", filed on December 25, 2022. Technical Field

[0002] This invention relates to the field of industrial microbiology, specifically to an actinomycete that uses glycerol as the sole carbon source to ferment and produce metabolites containing glycerol groups, and more specifically to Micromonospora deltaeniae and its applications in industry, agriculture and medicine. Background Technology

[0003] Microorganisms can utilize glycerol to produce fine chemicals, with *Klebsiella pneumoniae*, *Citrobacter flexneri*, and *Clostridium butyricum* being the most studied (Sun YQ, Shen JT, Yan L, et al. Advances in bioconversion of glycerol to 1,3-propanediol: prospects and challenges[J]. Process Biochemistry, 2018, 71: 134-146.). In recent years, the use of various microorganisms to convert crude glycerol into fine chemicals has attracted attention from scholars both domestically and internationally. Qi Xianghui et al. provided a microbial co-culture system for producing 1,3-propanediol via glycerol fermentation, which improved the conversion rate of glycerol and the yield of 1,3-propanediol while reducing production costs (CN111394396B). Zhao Zongbao et al. used this method in extracellular cell-free systems or in microbial in vivo catalysis of glycerol, a byproduct of biodiesel, to produce 1,3-propanediol, significantly reducing the production cost of 1,3-propanediol and increasing the product yield (CN110964753B).

[0004] Glycerol is an unavoidable byproduct in the biodiesel transesterification process, and its production has increased dramatically with the development of the biodiesel industry. Microbial conversion has the advantages of mild conditions, simplicity, and ease of operation, which has led to widespread attention on the application of glycerol in the fermentation field (Chilakamarry CR, Sakinah AM, Zularisam AW, et al. Bioconversion of glycerol into biofuels—opportunities and challenges[J]. BioEnergy Research, 2022, 15(1):46-61.). Utilizing microbial cell factories to convert renewable biomass resources into fine chemicals has important industrial, agricultural, and pharmaceutical application value (Jathanna HM, Rao C V. Using Aspergillus ochraceus, a Native Fungus, to Convert Biodiesel-Derived Crude Glycerol to Single Cell Oil for Commercial Applications[J]. Waste and Biomass Valorization, 2022, 13(6):2831-2845.). Summary of the Invention

[0005] This invention isolated a strain of *Micromonospora profundi* TRM95458 from the rhizosphere soil of chickpeas in Mulei County, and investigated its secondary metabolites. It was discovered that this strain can produce a novel compound ABAGG (i.e., compound B) and compounds A, D, and E using glycerol as the sole carbon source. The structural formulas are as follows:

[0006]

[0007] Therefore, the technical problem to be solved by the present invention is to provide a micromonospora that produces ABAGG, namely Micromonospora profundi TRM95458, which was deposited on August 2, 2022 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) (address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology), with accession number GDMCC NO:62600 and classification name: Micromonospora profundi. It has been tested and found to be viable.

[0008] The application of Micromonospora prolifera in the conversion of glycerol and glycine is characterized by its use of glycerol as the sole carbon source to ferment and produce metabolites containing glycerol groups, which are used to convert renewable biomass resources into fine chemicals for industrial, agricultural and pharmaceutical applications.

[0009] This invention provides the application of the aforementioned *Micromonospora* in the production of ABAGG. The application is characterized by the following steps: culturing the *Micromonospora* under suitable conditions to obtain a fermentation broth; separating and preparing ABAGG from the fermentation broth, wherein the fermentation medium for culturing the actinomycetes uses glycerol as the sole carbon source and glycine, etc., as nitrogen sources.

[0010] The culture medium formulation and cultivation conditions for the fermentation of *Micromonas hydrophila* are as follows: Seed culture medium: starch 10g, TSB 3g, glucose 4g, yeast extract 4g, malt extract 10g, trace elements 1mL, agar 17g, pH adjusted to 7.0. Solid plate mycelium (1cm in diameter) is inoculated into 500mL of seed culture medium (150mL volume, 3-5 mycelium cakes per bottle), and cultured at 30℃ and 180rpm for 5 days. Fermentation broth culture medium: glycerol 20mL, peptone 10g, glycine 10g, yeast extract 10g, trace element solution 1mL, pH adjusted to 7.0. 4% of this is inoculated into the fermentation medium, and cultured on a shaker at 30℃ and 180rpm for 7 days.

[0011] The method for separating and preparing ABAGG from the fermentation broth is as follows: after centrifugation, the fermentation broth is wet-loaded onto a D101 macroporous resin column, and then impurities such as sugars, salts and water-soluble proteins are removed with 4 column volumes of distilled water. After elution with 30% to 80% methanol, the 30% to 80% methanol eluents are combined and concentrated under reduced pressure using a rotary evaporator to obtain the crude extract of the fermentation broth.

[0012] The method for separating and purifying ABAGG from fermentation broth further includes the following steps: drying the crude extract of fermentation broth, dissolving it in methanol, passing it through an ODS column, eluting it with 40%, 60%, and 80% methanol respectively, collecting the 80% methanol eluent and concentrating it into an extract, and continuing to prepare ABAGG by pHPLC.

[0013] The pHPLC preparation conditions were as follows: A: 30% MeOH-H2O (0.075% HCOOH); B: 100% MeOH; flow rate: 10 mL / min; gradient elution conditions: 0-40 min 0-65% B; 40-45 min 65-100% B; 45-50 min 100% B; DAD detector wavelength: 350 nm; and the peak was collected at a retention time RT = 21.85 min to obtain ABAGG.

[0014] This invention also provides a compound ABAGG, which has the effect of regulating cell osmotic pressure and can improve the salt tolerance of organisms. Its structural formula is as follows:

[0015]

[0016] Furthermore, the present invention also provides the application of the compound ABAGG in regulating cell osmotic pressure or improving biological salt tolerance.

[0017] The present invention has the following beneficial effects: Micromonospora adenophora TRM95458 can produce metabolites containing glycerol groups using glycerol as the sole carbon source, which can be used to convert renewable biomass resources into fine chemicals for application in industry, agriculture and medicine. Attached Figure Description

[0018] Figure 1 Scanning electron microscope image of Micromonospora deepis TRM95458 (showing mycelium and round spores).

[0019] Figure 2 Taxonomic position and phylogenetic tree of Micromonospora deepis TRM95458.

[0020] Figure 3 High-performance liquid chromatography (HPLC) chromatogram of fermentation products prepared from strain 95458.

[0021] Figure 4 Two-dimensional nuclear magnetic resonance HMBC long-range correlation diagram of ABAGG.

[0022] Figure 5 The proton NMR spectrum of ABAGG.

[0023] Figure 6 The carbon NMR spectrum of ABAGG.

[0024] Figure 7 The nuclear magnetic resonance HMBC spectrum of ABAGG.

[0025] Figure 8 HPLC quantitative analysis standard curve of ABAGG.

[0026] Figure 9 The yield of ABAGG produced by Micromonospora deepis TRM95458 with different amounts of added glycerol.

[0027] Figure 10 The yield of ABAGG produced by Micromonospora deepis TRM95458 with different amounts of glycine addition.

[0028] Figure 11The relationship between salt stress and the accumulation of ABAGG osmotic substances in Micromonospora filamentosa TRM95458. Detailed Implementation

[0029] The present invention will be further illustrated below through a detailed description of specific embodiments, but this is not intended to limit the invention and is merely an illustrative example.

[0030] Example 1: Isolation and Classification Identification of Micromonospora deepis TRM95458

[0031] 1. Micromonospora prolifera TRM95458 isolated

[0032] Actinomycetes were isolated from the rhizosphere soil samples (0-30 cm) of chickpeas in Mulei County, Xinjiang, using the dilution plating method. The samples were cultured on 1 / 10 ISP2 medium (4 g glucose, 4 g yeast extract, 10 g malt extract, 1 mL trace elements, 17 g agar, pH adjusted to 7.0) at 28°C. Further screening with glycerol-amino acid composite medium yielded strain TRM 95458.

[0033] 2. Classification and identification of Micromonospora filamentosa TRM95458

[0034] 2.1 Morphological observation

[0035] Electron microscopy observation: For solid plate culture, cut off a piece of mycelium (approximately 0.5cm × 0.5cm) with a blade, removing excess culture medium. The culture medium containing mycelium should be thin and uniform in thickness. Observe and record the mycelial morphology, aerial hyphae, and intra-molecular hyphae growth using an electron microscope, noting whether the mycelium produces spore hyphae and their arrangement and shape; spore shape and size, etc. Scanning electron microscope images are shown below. Figure 1 .

[0036] Micromonas hydrophila TRM95458 grew well on ISP2 medium. The colonies were round and flat with a dry surface, neat edges, abundant aerial hyphae, and yellow hyphae with yellowish-brown soluble pigment.

[0037] 2.2 Molecular biological identification of strain TRM95458

[0038] 2.2.1 Method for extracting genomic DNA from strain TRM95458

[0039] Collect TRM95458 bacterial cells from the culture plate and place them in a 1.5 mL sterile centrifuge tube. Add 480 μL of 1×TE buffer. Add 20 μL of lysozyme (50 mg·mL⁻¹). -1 Place in a 37°C water bath overnight. Add 50 μL of 20% SDS and 5 μL of 20 mg / mL solution to each tube. -1Proteinase K was added and incubated in a water bath at 60°C for 2 hours. Then, 550 μL of a phenol:chloroform:isoamyl alcohol solution (25:24:1) was added, and the mixture was centrifuged at 12000 rpm for 5 minutes. The supernatant was transferred to another centrifuge tube, and the extraction was repeated twice. The supernatant was then collected, and 300 μL of 95% isopropanol and 70 μL of sodium acetate (3 mol·L⁻¹) were added. -1 Centrifuge at 12000 rpm for 10 min and discard the supernatant. Wash the centrifuged product once with 500 μL of 70% ethanol, centrifuge at 12000 rpm for 5 min, discard the supernatant, and allow the ethanol to evaporate completely. Dissolve the DNA at the bottom thoroughly with 30 μL of sterile ultrapure water. Assess the DNA extraction quality by 1% agarose gel electrophoresis. Store the extracted DNA at -20°C for later use.

[0040] 2.2.2 Method for amplifying the 16S rRNA gene of strain TRM95458

[0041] The 16S rRNA gene fragment in the genomic DNA of actinomycetes was amplified using universal primers 27F (5'-AGAGTTTGATCCTGGCTC-3') and 1492R (5'-CGGCTACCTTGTTACGACTT-3'). The PCR reaction mixture consisted of 34 μL dd H2O and 10× Buffer (containing Mg2+). 2+ 5 μL, dNTPs 2.5 μL, primer 27F (10 μmol·L⁻¹) -1 2 μL, primer 1492R (10 μmol·L⁻¹) -1 2 μL of 50% DMSO, 0.5 μL of Taq DNA polymerase, and 2 μL of template DNA.

[0042] The PCR reaction conditions were as follows: pre-denaturation at 94℃ for 4 min; denaturation at 94℃ for 1 min, annealing at 56℃ for 1 min, extension at 72℃ for 2 min, 30 cycles; total extension at 72℃ for 8 min. After the reaction, the results were detected by 1% agarose gel electrophoresis. The PCR products that met the conditions were then sequenced.

[0043] 2.2.3 Comparison and analysis methods for sequencing results

[0044] Sequencing results were assembled using DNAMAN 5.2 software. The sequences were compared with published strain sequences in the GenBank database using BLAST. The 16S rDNA gene sequences of published strains with high similarity were downloaded. A phylogenetic tree was constructed using MEGA 5.0 software to determine the taxonomic position of strain TRM95458.

[0045] 2.2.4 Results of 16S rDNA sequencing of strain TRM95458

[0046] The sequencing results were assembled using DNAMAN 5.2 software, confirming that the fragment consisted of 1381 bases, yielding the 16S rDNA sequence of TRM95458 as follows:

[0047]

[0048]

[0049] 2.2.5 Construction of Homologous Evolutionary Tree

[0050] BLAST alignment was used to download sequences with close similarity to the 16S rDNA gene sequence of the target actinomycete from the GenBank database. Multiple sequence alignment of the actinomycete 16S rDNA gene sequence was performed using MEGA 5.0 software, and a phylogenetic tree was constructed (see [link to phylogenetic tree]). Figure 2 The 16S rDNA gene sequence of strain TRM95458 and that of *Micromonospora profundi* (with 100.00% identity) clustered on the same phylogenetic branch, therefore the micromonospora was identified as *Micromonospora profundi* TRM95458.

[0051] Example 3: Fermentation and preparation of ABAGG using Micromonospora deepis TRM95458

[0052] A small amount of mycelium was inoculated into ISP2 medium and cultured at 28℃ for 5 days to obtain the seed culture of *Micromonospora deliciosa* TRM95458. The seed culture (4% inoculum) was then inoculated into fermentation medium for fermentation. The fermentation conditions were: 180 rpm, initial pH 7.0-7.2, 150 mL volume, 30℃, and fermentation for 7 days. The fermentation broth was collected, centrifuged, and the supernatant was wet-loaded onto a macroporous resin column (each 100 L of fermentation filtrate was packed with 2.0 kg of D101). The column was then eluted with 4 column volumes of water, 30% methanol, 80% methanol, and 95% methanol, respectively. After concentration under reduced pressure to remove the solvent, a methanol extract of the fermentation broth rich in *Micromonospora deliciosa* was obtained. The methanol-dissolved extract was passed through an ODS column and eluted with 20%, 40%, 60%, and 80% methanol, respectively. The eluent was collected and analyzed by HPLC. Collect 80% of the methanol eluent and perform preparative liquid chromatography separation and preparation (see...). Figure 3 The collected samples were analyzed using 1D & 2D NMR techniques to determine the structure of the compounds, yielding a new compound, ABAGG. The HMBC relationship diagram, 1H NMR spectrum, 1C NMR spectrum, and HMBC spectrum of the new compound ABAGG are shown below. Figure 4 , Figure 5 , Figure 6 and Figure 7 The 1H and 1C NMR data of the ABAGG series are shown in Table 1.

[0053] Table 1. ABAGG NMR spectral data (H1 and C1)

[0054]

[0055]

[0056] Example 4: Quantitative Analysis and Structural Characterization of ABAGG

[0057] ABAGG was quantitatively analyzed by high-performance liquid chromatography (HPLC). The chromatographic conditions were: a 4.6 mm × 150 mm C18 column (0.5 μm), 80% methanol-water as the mobile phase, a flow rate of 1.0 mL / min, and detection wavelengths of 254 nm, 280 nm, 330 nm, and 445 nm. The structures of the four compounds obtained are as follows:

[0058]

[0059] Among them, compound B (i.e., ABAGG) is a compound with a new structure.

[0060] Example 5: Determination of the glycerol conversion function of Micromonospora prolifera TRM95458 in the fermentation production of ABAGG

[0061] 1. The purified ABAGG product was prepared into a 1.0 mg / mL stock solution, and then quantitatively diluted to a series of concentrations of 1.0, 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0.0156 mg / mL. The standard curve was determined by HPLC using the area normalization method (see [link to standard curve]). Figure 8 The ABAGG standard curve equation is: y = 4.2905x + 0.3875, where x is the relative peak area and y is the ABAGG content. According to R... 2 =0.9929 indicates a good linear relationship, which can be used to determine the content of ABAGG.

[0062] 2. Different amounts of glycerol and glycine were added to the fermentation broth culture medium (10g millet, 100g bean sprouts, 10g glucose, 5g peptone, 2.5g NaCl, 1g (NH4)2SO4, pH adjusted to 7.0) to obtain different amounts of ABAGG. The results showed that the highest ABAGG yield was achieved when 0.1mL of glycerol and 1g of glycine were added. Figure 9 The output of ABAGG produced by Micromonospora deepis TRM95458 was shown under different glycerol addition levels; Figure 10This shows the yield of ABAGG produced by Micromonospora deepis TRM95458 when different amounts of glycine were added.

[0063] Example 6: Relationship between salt stress and the accumulation of ABAGG osmotic substances in Micromonospora salina TRM95458

[0064] Adding 0 g / L, 2.5 g / L, 5 g / L, 10 g / L, 15 g / L, and 20 g / L NaCl to the fermentation broth of Micromonospora deepis TRM95458 resulted in an initial increase followed by a decrease in ABAGG production as the NaCl concentration increased. The highest ABAGG production was observed when the NaCl concentration was 10 g / L. Figure 11 (As shown). This indicates that at higher salt concentrations, *Micromonospora deepis* TRM95458 enhances its salt tolerance by converting glycerol and glycine and accumulating the osmoregulating substance ABAGG intracellularly. ABAGG is a novel, osmoregulating, and compatible substance.

Claims

1. Application of *Micromonospora profundi* TRM95458 in the conversion of glycerol and glycine, wherein the accession number of *Micromonospora profundi* TRM95458 is GDMCC NO: 62600, and the conversion produces compounds with the following structures: 。 2. A method for converting glycerol and glycine, characterized in that, Glycerol and glycine were converted by fermentation of *Micromonospora profundi* TRM95458 in a culture medium using glycerol as the sole carbon source and glycine as the nitrogen source; wherein the accession number of *Micromonospora profundi* TRM95458 is GDMCC NO:62600; the conversion produced a compound with the following structure: 。

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